Enzymes are widely used as labels in immunoassays and DNA probe assays. In order to achieve good performance, the enzyme must be stable, highly reactive, available in high specific activity, easy to conjugate to other proteins or haptens and safe to use. In addition, a suitable substrate for the enzyme must be readily available. The requirements of a suitable substrate include high sensitivity of the reaction product (i.e., high extinction coefficient or high fluorescence), low background signal (i.e., low starting absorbance or low starting fluorescence) and good stability (i.e., substrate does not decompose to give chromophore or fluorophore absorbing at the measurement wave length unless enzyme is present). These requirements are particularly important for automated assays which require minimum calibration frequency.
The stability of an enzyme substrate can be thought of in terms of the amount of time that the substrate can be used as a substrate in a particular enzyme assay. Factors that influence the useful lifetime of a substrate include the chemical stability of the substrate in the milieu it is stored in, the amount of background signal (i.e., fluorescence, absorbance, etc.) present in the substrate preparation and the amount of signal being detected in the enzyme assay.
Substrate background is the signal generated from the substrate degradation products. These degradation products can result from impurities generated during the synthesis and purification of the substrate, from breakdown of the substrate (instability) during storage or from product formed during an assay in the absence of the enzyme. Although some background signal from the substrate can be tolerated, high background signals relative to the specific signal being detected in the assay will result in poor assay performance.
Poor substrate stability will result in using suboptimal substrate concentrations since the initial substrate concentration (and background signal) must be low enough to allow for the continuously increasing background signal caused by the instability. The high, changing background will require more frequent instrument blanking and make accurate measure of enzyme activity more difficult.
Among many commonly used fluorescent enzyme substrates, 4-methyl umbelliferyl esters offer high sensitivity and are readily available for many enzymes. 4-Methyl umbelliferyl esters are fluorescent but at a shorter wavelength than the hydrolyzed product, i.e., 4-methyl umbelliferone. Thus, the spectral interference can be minimized by selecting the measurement wavelength optimal for the 4-methyl umbelliferone product. However, the instability and high background fluorescence of methyl umbelliferyl esters have limited their wide application in commercial products. Methyl umbelliferyl esters are part of a larger class of compounds known as hydroxy coumarin esters.
B. N. Mattoo (Trans. Faraday Soc., 1957, 53, 760-766) describes the structural changes of coumarin at high pH, but the reverse reaction is not discussed. Mattoo does not discuss the stability of coumarin or of hydroxy coumarin esters.
In Trans. Faraday Soc., 1958, 54, 19-24, Mattoo discusses the dissociation constants of hydroxy coumarins. Mattoo does not discuss the stability of hydroxy coumarins or the reduction of their fluorescence in high concentrations of base.
Fernley et al., in Biochem. J., Vol. 97, 95-103 (1965), discuss the use of 4-methyl umbelliferyl phosphate (MUP) as a substrate for the measurement of calf intestinal phosphatase activities. They detail the kinetic behavior of the enzyme as a function of pH, ionic strength and temperature. The MUP was dissolved in 0.5M K.sub.2 HOP.sub.4 -KOH buffer, pH 10.4, and used in the phosphatase assay at various pH values in the range of 7.55 to 10.4.
In Analytical Chemistry, 42, 990-993 (1970), Fink et al. discuss the effect of pH on the fluorescence properties of umbelliferone. Fink et al. do not use pH values higher than 12.7 (0.05N NaOH) and they do not discuss the stability of umbelliferyl esters.
In U.S. Pat. No. 3,466,306, issued Sep. 9, 1969 Babson et al. describe the stabilization of certain phosphate ester substrates in an alkaline buffer of pH 9.7 to 10.5. Babson et al. do not mention 4-methyl umbelliferyl phosphate as a compound to be stabilized by their method. Also, while the method of Babson et al. slows down the hydrolysis of phosphate esters, it does not make long-term storage of 4-methyl umbelliferyl phosphate feasible. Because of the high fluorescence sensitivity of 4-methyl umbelliferone, a trace hydrolysis of 4-methyl umbelliferyl phosphate will result in an unacceptably high background signal.
There still exists a need for compositions and methods which will provide a very stable storage milieu for hydroxy coumarin esters in general and methyl umbelliferyl esters specifically while at the same time providing for a reduction in the background fluorescence due to contaminating hydrolysis products. This invention provides such compositions and methods.